Technical Notes

Mitochondria are found in eukaryotic cells, where they make up as much as 10% of the cell volume. They are pleomorphic organelles with structural variations depending on cell type, cell-cycle stage and intracellular metabolic state. The key function of mitochondria is energy production through oxidative phosphorylation (OxPhos) and lipid oxidation. Several other metabolic functions are performed by mitochondria, including urea production and heme, non-heme iron and steroid biogenesis, as well as intracellular Ca2+ homeostasis. Mitochondria also play a pivotal role in apoptosis—the genetically controlled ablation of cells during normal development (Assays for Apoptosis—Section 15.5). For many of these mitochondrial functions, there is only a partial understanding of the components involved, with even less information on mechanism and regulation.

The morphology of mitochondria is highly variable. In dividing cells, the organelle can switch between a fragmented morphology with many ovoid-shaped mitochondria, as is often shown in textbooks, and a reticulum in which the organelle is a single, many-branched structure. The cell cycle– and metabolic state–dependent changes in mitochondrial morphology are controlled by a set of proteins that cause fission and fusion of the organelle mass. Mutations in these proteins are the cause of several human diseases, indicating the importance of overall morphology for cell functioning (Mitochondria in Diseases—Note 12.2). Organelle morphology is also controlled by cytoskeletal elements, including actin filaments and microtubules (Figure 12.2.1). In nondividing tissue, overall mitochondrial morphology is very cell-type dependent, with mitochondria spiraling around the axoneme in spermatozoa, and ovoid bands of mitochondria intercalating between actomyosin filaments. There is evidence of functionally significant heterogeneity of mitochondrial forms within individual cells.

The abundance of mitochondria varies with cellular energy level and is a function of cell type, cell-cycle stage and proliferative state. For example, brown adipose tissue cells, hepatocytes and certain renal epithelial cells tend to be rich in active mitochondria, whereas quiescent immune-system progenitor or precursor cells show little staining with mitochondrion-selective dyes. The number of mitochondria is reduced in Alzheimer disease and their proteins and nucleic acids are susceptible to damage by reactive oxygen species, including nitric oxide (Probes for Reactive Oxygen Species, Including Nitric Oxide—Chapter 18).

We provide a range of mitochondrion-selective fluorescent proteins and organic dyes with which to monitor mitochondrial morphology and organelle functioning. In contrast to the fluorescent protein–based CellLight probes, the uptake of most mitochondrion-selective organic dyes is dependent on the mitochondrial membrane potential. These dyes thereby enable researchers to probe mitochondrial activity, localization and abundance, as well as to monitor the effects of some pharmacological agents that alter mitochondrial function. The CellLight probes can be used in combination with these organic dyes to investigate relationships between mitochondrial morphology and membrane potential.

Mitochondrial motility during mitosis can be easily observed in cells transduced with CellLight Mitochondria-GFP or CellLight Mitochondria-RFP (Figure 12.2.3). In contrast to MitoTracker Red CMXRos, TMRE, rhodamine 123 and other cationic dyes, mitochondrial localization of fluorescent protein–based markers is not driven by membrane potential. They can therefore be used in combination with cationic dye probes to investigate relationships between mitochondrial morphology and membrane potential.

Figure 12.2.3 Mitochondrial dynamics during mitosis. U2OS cells were transduced with CellLight® Mitochondria-RFP reagent (C10505, C10601) and imaged every 5 min for 16 hr. Extensive mitochondrial motility is seen throughout mitosis and following mitosis, as the cell regains its pre-mitotic shape.

MitoTracker Probes: Fixable Mitochondrion-Selective Probes

Although conventional fluorescent stains for mitochondria, such as rhodamine 123 and tetramethylrosamine, are readily sequestered by functioning mitochondria, they are subsequently washed out of the cells once the mitochondrion's membrane potential is lost. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria. To overcome this limitation, we have developed MitoTracker probes—a series of mitochondrion-selective stains that are concentrated by active mitochondria and well retained during cell fixation. Because the MitoTracker Orange, MitoTracker Red and MitoTracker Deep Red probes are also retained following permeabilization, the sample retains the fluorescent staining pattern characteristic of live cells during subsequent processing steps for immunocytochemistry, in situ hybridization or electron microscopy. In addition, MitoTracker reagents eliminate some of the difficulties of working with pathogenic cells because, once the mitochondria are stained, the cells can be treated with fixatives before the sample is analyzed.

Properties of MitoTracker Probes

MitoTracker probes are cell-permeant mitochondrion-selective dyes that contain a mildly thiol-reactive chloromethyl moiety. The chloromethyl group appears to be responsible for keeping the dye associated with the mitochondria after fixation. To label mitochondria, cells are simply incubated in submicromolar concentrations of the MitoTracker probe, which passively diffuses across the plasma membrane and accumulates in active mitochondria. Once their mitochondria are labeled, the cells can be treated with aldehyde-based fixatives to allow further processing of the sample; with the exception of MitoTracker Green FM, subsequent permeabilization with cold acetone does not appear to disturb the staining pattern of the MitoTracker dyes.

Figure 12.2.4 Intracellular reactions of our fixable, mitochondrion-selective MitoTracker Orange CM-H2TMRos (M7511). When this cell-permeant probe enters an actively respiring cell, it is oxidized to MitoTracker Orange CMTMRos and sequestered in the mitochondria, where it can react with thiols on proteins and peptides to form aldehyde-fixable conjugates.

Figure 12.2.5 Flow cytometric analysis of Jurkat cells using the Mitochondrial Membrane Potential/Annexin V Apoptosis Kit (V35116). Jurkat human T-cell leukemia cells in complete medium were A) first exposed to 10 µM camptothecin for 4 hours or B) left untreated. Both cell populations were then treated with the reagents in the Mitochondrial Membrane Potential/Annexin V Apoptosis Kit and analyzed by flow cytometry. Note that the apoptotic cells show higher reactivity for annexin V and lower MitoTracker Red dye fluorescence than do live cells.

MitoTracker Green FM Dye

Mitochondria in cells stained with nanomolar concentrations of MitoTracker Green FM dye (M7514, ) exhibit bright green, fluorescein-like fluorescence (, , ). The MitoTracker Green FM probe has the added advantage that it is essentially nonfluorescent in aqueous solutions and only becomes fluorescent once it accumulates in the lipid environment of mitochondria. Hence, background fluorescence is negligible, enabling researchers to clearly visualize mitochondria in live cells immediately following addition of the stain, without a wash step.

Unlike MitoTracker Orange CMTMRos and MitoTracker Red CMXRos, the MitoTracker Green FM probe appears to preferentially accumulate in mitochondria regardless of mitochondrial membrane potential in certain cell types, making it a possible tool for determining mitochondrial mass. Furthermore, the MitoTracker Green FM dye is substantially more photostable than the widely used rhodamine 123 fluorescent dye and produces a brighter, more mitochondrion-selective signal at lower concentrations. Because its emission maximum is blue-shifted approximately 10 nm relative to the emission maximum of rhodamine 123, the MitoTracker Green FM dye produces a fluorescent staining pattern that should be better resolved from that of red-fluorescent probes in double-labeling experiments. The mitochondrial proteins that are selectively labeled by the MitoTracker Green FM reagent have been separated by capillary electrophoresis.

Image-iT LIVE Mitochondrial and Nuclear Labeling Kit

The Image-iT LIVE Mitochondrial and Nuclear Labeling Kit (I34154) provides two stains—red-fluorescent MitoTracker Red CMXRos dye (excitation/emission maxima ~578/599 nm) and blue-fluorescent Hoechst 33342 dye (excitation/emission maxima when bound to DNA ~350/461 nm)—for highly selective mitochondrial and nuclear staining, respectively, in live, GFP–transfected cells. These dyes can be combined into one staining solution using the protocol provided, saving labeling time and wash steps while still providing optimal staining. Both dyes are retained after formaldehyde fixation and permeabilization. The Image-iT LIVE Mitochondrial and Nuclear Labeling Kit contains:

MitoSOX Red Mitochondrial Superoxide Indicator

Mitochondrial superoxide is generated as a by-product of oxidative phosphorylation. In an otherwise tightly coupled electron transport chain, approximately 1–3% of mitochondrial oxygen consumed is incompletely reduced; these "leaky" electrons can quickly interact with molecular oxygen to form superoxide anion, the predominant reactive oxygen species in mitochondria. Increases in cellular superoxide production have been implicated in cardiovascular diseases, including hypertension, atherosclerosis and diabetes-associated vascular injuries, as well as in neurodegenerative diseases such as Parkinson disease, Alzheimer disease and amyotrophic lateral sclerosis (ALS).

MitoSOX Red mitochondrial superoxide indicator (M36008) is a cationic derivative of dihydroethidum (also known as hydroethidine; see below) designed for highly selective detection of superoxide in the mitochondria of live cells (). The cationic triphenylphosphonium substituent of MitoSOX Red indicator is responsible for the electrophoretically driven uptake of the probe in actively respiring mitochondria. Oxidation of MitoSOX Red indicator (or dihydroethidium) by superoxide results in hydroxylation at the 2-position (Figure 12.2.6). 2-Hydroxyethidium (and the corresponding derivative of MitoSOX Red indicator) exhibit a fluorescence excitation peak at ~400 nm that is absent in the excitation spectrum of the ethidium oxidation product generated by reactive oxygen species other than superoxide. Thus, fluorescence excitation at 400 nm with emission detection at ~590 nm provides optimum discrimination of superoxide from other reactive oxygen species (Figure 12.2.7).

Measurements of mitochondrial superoxide generation using MitoSOX Red indicator in mouse cortical neurons expressing caspase-cleaved tau microtubule-associated proteinhave been correlated with readouts from fluorescent indicators of cytosolic and mitochondrial calcium and mitochondrial membrane potential. The relationship of mitochondrial superoxide generation to dopamine transporter activity, measured using the aminostyryl dye substrate 4-Di-1-ASP (D288, see below), has been investigated in mouse brain astrocytes. MitoSOX Red indicator has been used for confocal microscopy analysis of reactive oxygen species (ROS) production by mitochondrial NO synthase (mtNOS) in permeabilized cat ventricular myocytes and, in combination with Amplex Red reagent, for measurement of mitochondrial superoxide and hydrogen peroxide production in rat vascular endothelial cells. In addition to imaging and microscope photometry measurements, several flow cytometry applications of MitoSOX Red have also been reported. Detailed protocols for simultaneous measurements of mitochondrial superoxide generation and apoptotic markers APC annexin V (A35110, Assays for Apoptosis—Section 15.5) and SYTOX Green (S7020, Nucleic Acid Stains—Section 8.1) in human coronary artery endothelial cells by flow cytometry have been published by Mukhopadhyay and co-workers.

Figure 12.2.7 Selectivity of the MitoSOX Red mitochondrial superoxide indicator (M36008). Cell-free systems were used to generate a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS); each oxidant was then added to a separate 10 µM solution of MitoSOX Red reagent and incubated at 37°C for 10 minutes. Excess DNA was added (unless otherwise noted) and the samples were incubated for an additional 15 minutes at 37°C before fluorescence was measured. The Griess Reagent Kit (G7921) (for nitric oxide, peroxynitrite, and nitrite standards only; blue bars) and dihydrorhodamine 123 (DHR 123, (D632); green bars) were employed as positive controls for oxidant generation. Superoxide dismutase (SOD), a superoxide scavenger, was used as a negative control for superoxide. The results show that the MitoSOX Red probe (red bars) is readily oxidized by superoxide but not by the other oxidants.

RedoxSensor Red CC-1 Stain

RedoxSensor Red CC-1 stain (2,3,4,5,6-pentafluorotetramethyldihydrorosamine, R14060; ) passively enters live cells and is subsequently oxidized in the cytosol to a red-fluorescent product (excitation/emission maxima ~540/600 nm), which then accumulates in the mitochondria. Alternatively, this nonfluorescent probe may be transported to the lysosomes where it is oxidized. The differential distribution of the oxidized product between mitochondria and lysosomes appears to depend on the redox potential of the cytosol. In proliferating cells, mitochondrial staining predominates; whereas in contact-inhibited cells, the staining is primarily lysosomal ().

JC-1 and JC-9: Dual-Emission Potential-Sensitive Probes

The green-fluorescent JC-1 probe (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide, T3168; ) exists as a monomer at low concentrations or at low membrane potential. However, at higher concentrations (aqueous solutions above 0.1 µM) or higher potentials, JC-1 forms red-fluorescent "J-aggregates" that exhibit a broad excitation spectrum and an emission maximum at ~590 nm (, , ). Thus, the emission of this cyanine dye can be used as a sensitive measure of mitochondrial membrane potential. Various types of ratio measurements are possible by combining signals from the green-fluorescent JC-1 monomer (absorption/emission maxima ~514/529 nm in water) and the J-aggregate (emission maximum 590 nm), which can be effectively excited anywhere between 485 nm and its absorption maximum at 585 nm (). The ratio of red-to-green JC-1 fluorescence is dependent only on the membrane potential and not on other factors that may influence single-component fluorescence signals, such as mitochondrial size, shape and density. Optical filters designed for fluorescein and tetramethylrhodamine can be used to separately visualize the monomer and J-aggregate forms, respectively. Alternatively, both forms can be observed simultaneously using a standard fluorescein longpass optical filter set. Chen and colleagues have used JC-1 to investigate mitochondrial potentials in live cells by ratiometric techniques (Figure 12.2.8).

Figure 12.2.8 Bivariate JC-1 (T3168) analysis of mitochondrial membrane potential in HL60 cells by flow cytometry. The sensitivity of this technique is demonstrated by the response to K+/valinomycin (V1644, Fluorescent Na+ and K+ Indicators—Section 21.1)–induced depolarization (panels B and D). Distinct populations of cells with different extents of mitochondrial depolarization are detectable following apoptosis-inducing treatment with 5 µM staurosporine for two hours (panel C). Figure courtesy of Andrea Cossarizza, University of Modena and Reggio Emilia, Italy.

Mitochondrion-Selective Rhodamines and Rosamines

Rhodamine 123

Rhodamine 123 (R302, R22420; ) is a cell-permeant, cationic, fluorescent dye that is readily sequestered by active mitochondria without inducing cytotoxic effects. Uptake and equilibration of rhodamine 123 is rapid (a few minutes) compared with dyes such as DASPMI (4-Di-1-ASP, D288), which may take 30 minutes or longer. Viewed through a fluorescein longpass optical filter, fluorescence of the mitochondria of cells stained by rhodamine 123 appears yellow-green. Viewed through a tetramethylrhodamine longpass optical filter, however, these same mitochondria appear red. Unlike the lipophilic rhodamine and carbocyanine dyes, rhodamine 123 apparently does not stain the endoplasmic reticulum.

Rosamines and Other Rhodamine Derivatives, Including TMRM and TMRE

Other mitochondrion-selective dyes include tetramethylrosamine (), whose fluorescence contrasts well with that of fluorescein for multicolor applications, and rhodamine 6G (R634, ), which has an absorption maximum between that of rhodamine 123 and tetramethylrosamine. Tetramethylrosamine and rhodamine 6G have both been used to examine the efficiency of P-glycoprotein–mediated exclusion from multidrug-resistant cells (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6). Rhodamine 6G has been employed to study microvascular reperfusion injury and the stimulation and inhibition of F1-ATPase from the thermophilic bacterium PS3.

The accumulation of tetramethylrhodamine methyl and ethyl esters (TMRM, T668; TMRE, T669) in mitochondria and the endoplasmic reticulum has also been shown to be driven by their membrane potential (Slow-Response Probes—Section 22.3). Moreover, because of their reduced hydrophobic character, these probes exhibit potential-independent binding to cells that is 10 to 20 times lower than that seen with rhodamine 6G. Tetramethylrhodamine ethyl ester has been described as one of the best fluorescent dyes for dynamic and in situ quantitative measurements—better than rhodamine 123—because it is rapidly and reversibly taken up by live cells. TMRM and TMRE have been used to measure mitochondrial depolarization related to cytosolic Ca2+ transients and to image time-dependent mitochondrial membrane potentials. A high-throughput assay utilizes TMRE and our low-affinity Ca2+ indicator fluo-5N AM (F14204, Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) to screen inhibitors of the opening of the mitochondrial transition pore.

Reduced Rhodamines and Rosamines

Inside live cells, the colorless dihydrorhodamines and dihydrotetramethylrosamine are oxidized to fluorescent products that stain mitochondria. However, the oxidation may occur in organelles other than the mitochondria. Dihydrorhodamine 123 (D632, D23806; ) reacts with hydrogen peroxide in the presence of peroxidases, iron or cytochrome c to form rhodamine 123. This reduced rhodamine has been used to monitor reactive oxygen intermediates in rat mast cells and to measure hydrogen peroxide in endothelial cells. Chloromethyl derivatives of reduced rosamines (MitoTracker Orange CM-H2TMRos, M7511; MitoTracker Red CM-H2XRos, M7513), which can be fixed in cells by aldehyde-based fixatives, have been described above.

Other Mitochondrion-Selective Probes

Carbocyanines

Most carbocyanine dyes with short (C1–C6) alkyl chains (Slow-Response Probes—Section 22.3) stain mitochondria of live cells when used at low concentrations (<100 nM); those with pentyl or hexyl substituents also stain the endoplasmic reticulum when used at higher concentrations (>1 µM). DiOC6(3) (D273) stains mitochondria in live yeast and other eukaryotic cells, as well as sarcoplasmic reticulum in beating heart cells. It has also been used to demonstrate mitochondria moving along microtubules. Photolysis of mitochondrion- or endoplasmic reticulum–bound DiOC6(3) specifically destroys the microtubules of cells without affecting actin stress fibers, producing a highly localized inhibition of intracellular organelle motility. We have included DiIC1(5) and DiOC2(3) in two of our MitoProbe Assay Kits for flow cytometry (M34151, M34150; Slow-Response Probes—Section 22.3). Several other potential-sensitive carbocyanine probes described in Slow-Response Probes—Section 22.3 also stain mitochondria in live cultured cells. The carbocyanine DiOC7(3) (D378), which exhibits spectra similar to those of fluorescein, is a versatile dye that has been reported to be a sensitive probe for mitochondria in plant cells.

Styryl Dyes

The styryl dyes DASPMI (4-Di-1-ASP, D288) and DASPEI (D426) can be used to stain mitochondria in live cells and tissues. These dyes have large fluorescence Stokes shifts and are taken up relatively slowly as a function of membrane potential. The kinetics of mitochondrial staining with styrylpyridinium dyes has been investigated using the concentration jump method.

Nonyl Acridine Orange

Nonyl acridine orange (A1372) is well retained in the mitochondria of live HeLa cells for up to 10 days, making it a useful probe for following mitochondria during isolation and after cell fusion. The mitochondrial uptake of this metachromatic dye is reported not to depend on membrane potential. It is toxic at high concentrations and apparently binds to cardiolipin in all mitochondria, regardless of their energetic state. This derivative has been used to analyze mitochondria by flow cytometry, to characterize multidrug resistance (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6) and to measure changes in mitochondrial mass during apoptosis in rat thymocytes.

Lucigenin

The well-known chemiluminescent probe lucigenin (L6868) accumulates in mitochondria of alveolar macrophages. Relatively high concentrations of the dye (~100 µM) are required to obtain fluorescent staining; however, low concentrations reportedly yield a chemiluminescent response to stimulated superoxide generation within the mitochondria. Molecular Probes lucigenin has been highly purified to remove a bright blue-fluorescent contaminant that is found in some commercial samples.

Mitochondrial Transition Pore Assays

The mitochondrial permeability transition pore, a nonspecific channel formed by components from the inner and outer mitochondrial membranes, appears to be involved in the release of mitochondrial components during apoptotic and necrotic cell death. In a healthy cell, the inner mitochondrial membrane is responsible for maintaining the electrochemical gradient that is essential for respiration and energy production. As Ca2+ is taken up and released by mitochondria, a low-conductance permeability transition pore appears to flicker between open and closed states. During cell death, the opening of the mitochondrial permeability transition pore dramatically alters the permeability of mitochondria. Continuous pore activation results from mitochondrial Ca2+ overload, oxidation of mitochondrial glutathione, increased levels of reactive oxygen species in mitochondria and other pro-apoptotic conditions. Cytochrome c release from mitochondria and loss of mitochondrial membrane potential are observed subsequent to continuous pore activation.

The Image-iT LIVE Mitochondrial Transition Pore Assay Kit (I35103), based on published experimentation for mitochondrial transition pore opening, permits a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. This assay employs the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl2, a quencher of calcein fluorescence, to selectively label mitochondria. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, calcein AM is cleaved by intracellular esterases to liberate the polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of CoCl2, while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl2 can also be treated with a Ca2+ ionophore such as ionomycin (I24222, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8) to allow entry of excess Ca2+ into the cells, which triggers mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence. This ionomycin response can be blocked with cyclosporine A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.

MitoProbe Transition Pore Assay Kit for Flow Cytometry

The MitoProbe Transition Pore Assay Kit (M34153), based on published experimentation for mitochondrial transition pore opening, permits a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone (Figure 12.2.9). As with the Image-iT LIVE mitochondrial transition pore assay described above, this assay employs the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl2, a quencher of calcein fluorescence, to selectively label mitochondria. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, calcein AM is cleaved by intracellular esterases to liberate the polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of CoCl2, while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl2 can also be treated with a Ca2+ ionophore such as ionomycin (I24222, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8) to allow entry of excess Ca2+ into the cells, which triggers mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence. This ionomycin response can be blocked with cyclosporine A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.

Figure 12.2.9 Flow cytometric analysis of Jurkat cells using the MitoProbe Transition Pore Assay Kit (M34153). Jurkat cells were incubated with the reagents in the MitoProbe Transition Pore Assay Kit and analyzed by flow cytometry. In the absence of CoCl2 and ionomycin, fluorescent calcein is present in the cytosol as well as the mitochondria, resulting in a bright signal (panel A). In the presence of CoCl2, calcein in the mitochondria emits a signal, but the cytosolic calcein fluorescence is quenched; the overall fluorescence is reduced, as compared with calcein alone (panel B). When ionomycin, a Ca2+ ionophore, and CoCl2 are added to the cells at the same time that calcein AM is added, the fluorescent signals from both the cytosol and mitochondria are largely abolished (panel C). The change in fluorescence between panels B and C indicates the continuous activation of mitochondrial permeability transition pores.

Yeast Mitochondrial Stain Sampler Kit

Because fluorescence microscopy has been extensively used to study yeast, we offer a Yeast Mitochondrial Stain Sampler Kit (Y7530). This kit contains sample quantities of five different probes that have been found to selectively label yeast mitochondria. Both well-characterized and proprietary mitochondrion-selective probes are provided:

Rhodamine 123

Rhodamine B hexyl ester ()

MitoTracker Green FM

SYTO 18 yeast mitochondrial stain

DiOC6(3)

The mitochondrion-selective nucleic acid stain included in this kit—SYTO 18 yeast mitochondrial stain—exhibits a pronounced fluorescence enhancement upon binding to nucleic acids, resulting in very low background fluorescence even in the presence of dye. SYTO 18 is an effective mitochondrial stain in live yeast but neither penetrates nor stains the mitochondria of higher eukaryotic cells. Each of the components of the Yeast Mitochondrial Stain Sampler Kit is also available separately, including the SYTO 18 yeast mitochondrial stain (S7529).

Abs and Em of styryl dyes are at shorter wavelengths in membrane environments than in reference solvents such as methanol. The difference is typically 20 nm for absorption and 80 nm for emission, but varies considerably from one dye to another. Styryl dyes are generally nonfluorescent in water.

This compound is susceptible to oxidation, especially in solution. Store solutions under argon or nitrogen. Oxidation may be induced by illumination.

This compound emits chemiluminescence at 470 nm upon oxidation in basic aqueous solutions.

The product generated by reaction of M36008 with superoxide has similar spectroscopic properties to ethidium bromide.

R14060 is colorless and nonfluorescent until oxidized. The spectral characteristics of the oxidation product (2,3,4,5,6-pentafluorotetramethylrosamine) are similar to those of MitoTracker Orange CMTMRos (M7510).

This product is specified to equal or exceed 98% analytical purity by HPLC.